Head and neck cancers are positive for human papillomavirus (HPV) in about 25% of the cases overall, and in up to 60% of the cases at specific sites, such as the oropharynx. A causal role of HPV (in particular HPV16) in the pathogenesis of oropharyngeal cancer (OPC) is now well accepted, as it is clear that HPV- positive cancers that are driven by the oncoproteins E6 and E7 constitute a disease of their own, with specific histological, molecular, clinical and epidemiological characteristics. HPV-positive tumors that exhibit E6/E7 expression (the surrogate marker for this is positive p16 staining) occur in younger patients; are more likely to be basaloid and relatively undifferentiated exhibit gene expression profiles in dicative of alterations of cell cycle and proliferation-related gene pathways; are not linked to alcohol consumption or smoking; and are linked to specific sexual behaviors. These tumors have a better prognosis than the HPV-negative tumors, which are epidemiologically linked to smoking and alcohol consumption and (according to our preliminary results, as well as other reports) exhibit gene expression profiles that are indicative of profound alterations of mechanisms of EMT, angiogenesis, and cell motility. We and others have shown that OPC from Black patients are often HPV-negative, while the HPV-positive cancers occur primarily in White patients. In addition, we have recently determined that HPV-positive OPC in Black patients are more often HPV-inactive: these tumors contain HPV DNA but do not express HPV oncoproteins and are p16-negative. We have shown that HPV-inactive tumors have gene expression profiles and survival rates similar to those of HPV-negative tumors. It is commonly assumed that in HPV-inactive tumors HPV is a passenger and has no role in their pathogenesis, however there are no studies corroborating or disputing this interpretation. We propose here the alternative and novel hypothesis that HPV-inactive tumors may start as HPV-driven lesions, at either pre- malignant or early invasive stages, and then turn to an HPV-inactive status by either mutational or epigenetic events. If our hypothesis is proven correct, this would represent a completely novel mechanism for HPV-mediated carcinogenesis that would cause us to re-think how HPV may cause cancer at sites other than the cervix. This application investigates the proposed hypothesis by two specific aims: 1) to analyze, by in situ methods of detection of HPV transcripts, the HPV expression status of a series of OPC cases positive for HPV-DNA in which the tumor mass is heterogeneous in terms of p16 expression; 2) to investigate potential mechanisms of escape from HPV control of growth in a model system for HPV16-mediated transformation in vitro. The results of these studies will clarify the role of HPV in OPC; allow for a more precise classification of OPC and HNC; and influence our evaluation of the effectiveness of HPV vaccines in the prevention of HNC. In addition, these results, if positive, would prompt a re-evaluation of the role of HPV at other cancer sites (i.e. lung, breast esophagus and rectum).